Accepted Manuscript Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line Benjamin Schmid, Kennie R. Prehn, Natakarn Nimsanor, Blanca Irene Aldana Garcia, Ulla Poulsen, Ida Jørring, Mikkel A. Rasmussen, Christian Clausen, Ulrike A. Mau-Holzmann, Sarayu Ramakrishna, Ravi Muddashetty, Rachel Steeg, Kevin Bruce, Peter Mackintosh, Andreas Ebneth, Bjørn Holst, Alfredo Cabrera- Socorro PII: S1873-5061(18)30279-4 DOI: https://doi.org/10.1016/j.scr.2018.11.010 Reference: SCR 1349 To appear in: Stem Cell Research Received date: 15 June 2017 Revised date: 5 November 2018 Accepted date: 19 November 2018 Please cite this article as: Benjamin Schmid, Kennie R. Prehn, Natakarn Nimsanor, Blanca Irene Aldana Garcia, Ulla Poulsen, Ida Jørring, Mikkel A. Rasmussen, Christian Clausen, Ulrike A. Mau-Holzmann, Sarayu Ramakrishna, Ravi Muddashetty, Rachel Steeg, Kevin Bruce, Peter Mackintosh, Andreas Ebneth, Bjørn Holst, Alfredo Cabrera-Socorro , Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line. Scr (2019), https://doi.org/10.1016/ j.scr.2018.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Accepted Manuscript
Generation of a set of isogenic, gene-edited iPSC lineshomozygous for all main APOE variants and an APOE knock-outline
Benjamin Schmid, Kennie R. Prehn, Natakarn Nimsanor, BlancaIrene Aldana Garcia, Ulla Poulsen, Ida Jørring, Mikkel A.Rasmussen, Christian Clausen, Ulrike A. Mau-Holzmann, SarayuRamakrishna, Ravi Muddashetty, Rachel Steeg, Kevin Bruce,Peter Mackintosh, Andreas Ebneth, Bjørn Holst, Alfredo Cabrera-Socorro
Received date: 15 June 2017Revised date: 5 November 2018Accepted date: 19 November 2018
Please cite this article as: Benjamin Schmid, Kennie R. Prehn, Natakarn Nimsanor, BlancaIrene Aldana Garcia, Ulla Poulsen, Ida Jørring, Mikkel A. Rasmussen, Christian Clausen,Ulrike A. Mau-Holzmann, Sarayu Ramakrishna, Ravi Muddashetty, Rachel Steeg, KevinBruce, Peter Mackintosh, Andreas Ebneth, Bjørn Holst, Alfredo Cabrera-Socorro ,Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOEvariants and an APOE knock-out line. Scr (2019), https://doi.org/10.1016/j.scr.2018.11.010
This is a PDF file of an unedited manuscript that has been accepted for publication. Asa service to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting proof beforeit is published in its final form. Please note that during the production process errors maybe discovered which could affect the content, and all legal disclaimers that apply to thejournal pertain.
Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE
knock-out line
Benjamin Schmida, Kennie R. Prehna, Natakarn Nimsanora, d, Blanca Irene Aldana Garciae, Ulla Poulsena, Ida Jørringa, Mikkel A. Rasmussena, Christian Clausena, Ulrike A. Mau-Holzmannc, Sarayu Ramakrishnag, h, Ravi Muddashettyg, Rachel Steegf, Kevin Brucef, Peter Mackintoshf, Andreas Ebneth b, Bjørn Holsta , Alfredo Cabrera-Socorrob a Bioneer A/S, Kogle Alle 2, 2970 Hørsholm, Denmark
b Janssen Research & Development, a division of Janssen Pharmaceutica; N.V., Neuroscience Therapeutic Area,
Turnhoutseweg 30, 2340 Beerse, Belgium
c Institute of Medical Genetics and Applied Genomics, Division of Cytogenetics, Calwerstrasse 7, Univ ersity of
Tuebingen, 72076, Germany
d Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Bangkok, 10700,
Thailand.
e Neurometabolism Research Unit, Department of Drug Design and Pharmacology, University of Copenhagen
f Censo Biotechnologies, Edinburgh, UK, EH25 9PP
g Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bengaluru, Karnataka, India
h University of Trans-Disciplinary Health Sciences & Technology (TDU), Bengaluru, Karnataka, India
Abstract
Alzheimer’s disease (AD) is the most frequent neurodegenerative disease amongst the elderly. The SNPs
rs429358 and rs7412 in the APOE gene are the most common risk factor for sporadic AD, and there are three
different alleles commonly referred to as APOE-ε2, APOE-ε3 and APOE-ε4. Induced pluripotent stem cells
(iPSCs) hold great promise to model AD as such cells can be differentiated in vitro to the required cell type.
Here we report the use of CRISPR/Cas9 technology employed on iPSCs from a healthy individual with an APOE-
ε3/ε4 genotype to obtain isogenic APOE-ε2/ε2, APOE-ε3/ε3, APOE-ε4/ε4 lines as well as an APOE-knock-out
line.
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cassette, and 1 µL of 100 µM ssODN using the P3 Primary Cell Kit (Lonza) using program CA167 following to the
manufacturer's instructions (Lonza). iPSCs were subsequently transferred back to a Matrigel-coated 100 mm
dish in E8 medium supplemented with 1:200 diluted Revita cell supplement (Gibco). 24 h post-nucleofection,
cells were subjected to puromycin (Invitrogen) selection for 4 hours at a concentration of 10 µg/mL and
allowed to recover for one week. Resistant colonies were then picked and expanded for genotyping.
Genotyping
DNA for genotyping was extracted using the prepgem kit from ZyGEM following the manufacturer’s
instructions. The DNA solution was diluted 1:5 with water. Genotyping was carried out using AmpliTaq Gold
Polymerase (Thermo Fisher) according to the manufacturer's instructions at an annealing temperature of 60° C
and the PCR primers APOE HhaI FW/RV (Table 3). The PCR products were digested using the restriction enzyme
HhaI (NEB) for 1h to detect genetically modified clones. Positive candidates were then sequenced using
sequencing primer APOE HhaI Seq FW (Table 3). Sequencing analysis of the KO line was carried out with the
PCR primers SURV APOE KO FW/RV and the sequencing primer SURV APOE KO seq FW (Table 3). Clones with a
frame shift were subjected to Western blot analysis. Briefly, iPSCs from one well of a 6-well plate were
detached with a cell scraper and transferred to a 2 mL Eppendorf tube and spun down at 120 g for 5 minutes.
The cell pellets were lysed in 50 µL of RIPA buffer (Invitrogen) containing Roche protease inhibitor. Lysates
were centrifuged at 14.000 g for 10 minutes at 4°C. The protein concentration was determined using the
Pierce BCA protein kit (Thermo Scientific) and 15 µg protein were loaded on an Invitrogen™ Novex™ pre-cast
Tris-Glycine 12% gel with trysglycine running buffer at 126V for 90 minutes and blotted on an Invitrogen™
Novex™ Nitrocellulose membrane at 35 V for 70 minutes. The membrane was blocked in 5% skim milk diluted
in TBS with 0.1% Tween 20 for 1 hour. Membrane was incubated with the ApoE antibody (NOVUS Biologicals,
NB110-60531, WUE-4, mouse, 1:1000) over night at room temperature. After washing, the blot was incubated
with goat anti-mouse IgG-HRP (sc-2005) from Santa Cruz Biotechnology (1:5000). Bands were visualized with
Pierce™ ECL Western Blotting Substrate (ThermoFisher).
Cell banking
iPSCs were grown in three 15 cm plates on matrigel in E8 medium to a density of 80%. Cells were detached
with 0.1% EDTA and centrifuged at 120g for 5 minutes. The cell pellet was resuspended in 50 mL of freezing
medium (50% E8, 40% FCS, 10% DMSO), and 1 mL aliquots were distributed in cryo vials. The vials were
transferred in isopropanol containers into a -80⁰ C freezer over night. For long term storage, the cells were
transferred into a nitrogen tank.
Morphology
The morphology was investigated by light microscopy 1 or 2 days after thawing one vial from the bank.
Karyotyping
For karyotyping, the cells were treated with colcimid (Gibco) when they were 60 – 80 % confluent. The cells
were then incubabted with 0.075 M KCl for 30 minutes at 37⁰ C and fixed with 1:3 acidic acid:methanol and
sent for G-band karyotyping (University of Tübingen). At least 15 metaphases were counted and 6 of them
were structurally evaluated by G-banding and a banding quality of 400-500.
STR Analysis
For the STR analysis, DNA was extracted (Qiagen) and analyzed using the AmpFLSTR Identifiler PCR
Amplification kit (Applied Biosystems).
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Microbiology
General microbiology was investigated by growing 500 µL of the supernatant in LB medium for 2 days at 37⁰ C.
Virology was investigated on the medium supernatant of the parental line by PCR analysis (Rasmussen et al.,
2014).
Integration of CRISPR plasmids
Analysis for the integration of CRISPR plasmid was carried out by PCR using primers U6-FW and pSpCas9-(339)-
RV1 (Table 3).
Expression of pluripotency markers:
Expression of pluripotency markers were investigated by both ICC and trilineage differentiation followed by
flow cytometry.
ICC:
iPSCs were grown on a glass coverslip coated with matrigel in E8 medium. When defined colonies were
detectable, the cells were fixed with ice cold methanol at -20 C⁰ C for 10 minutes. The cells were washed with
PBS and blocked with blocking solution (2% BSA and 0.1% Triton-X-100 in PBS) for 15 minutes at room
temperature. Primary antibodies were added in the respective dilution (Table 2) and incubated over night at 4⁰
C. The cells were washed three times with blocking solution and incubated with the respective secondary
antibody (Table 2) in blocking solution at room temperature for 1 hour. The cells were washed again three
times with blocking solution and once in water. The coverslips were finally put on glass slides with mounting
solution containing DAPI from Invitrogen and investigated by fluorescence microscopy.
Trilineage differentiation:
For trilineage differentiation, the iPSCs were split with accutase into a well of a 12-well plate with E8 medium
on matrigel in different densities: 200,000 cells/cm2 for ecto- and endoderm and 50,000 cells/cm2 for
mesoderm. For ectodermal differentiation, the medium was changed to neural induction medium (50% DMEM
F12 and 50% Neurobasal medium, 1X B27 without retinoic acid, 1X N2 supplement, 1X glutamax, 1X Pen/Strep
(all Gibco), 10 µM SB431542, 0.1 µM LDN193189 (both from Selleckchem)) on day one. The medium was
changed every day until day six. For endodermal differentiation, the medium was changed to MCDB131-1
medium (MCDB131 medium, 1.5 g/L NaHCO3, 1X glutamax, 1X Pen/Strep (all Gibco), 10 mM glucose (Sigma),
0.5% BSA) on day one including 3 µM CHIR99021 (Selleckchem) and 100ng/mL Activin A (Cell Gui dance
Systems). On day two, CHIR99021 was withdrawn and MCDB131-1 medium with activing A was changed every
day until day six. For mesodermal differentiation, the medium was replaced by mesodermal induction medium
(APEL medium (Gibco), 25 µg/mL Activin A (Cell Guidance Systems), 30 ng/mL BMP4 (Peprotech), 50 ng/mL
VEGF (peprotech), 1.5 µM CHIR99021 (Selleckchem)), which was left on the cells for two days. On day three,
the medium was changed to vascular specification medium (APEL medium, 50 ng/mL VEGF, 10 µM SB431542
(Selleckchem)), which was then changed every day until day six.
Flow cytometry:
After 6 days, all the trilineage differentiations were split with 0.5 mL accutase (10 minutes incubation at 37⁰ C).
The cells were mixed with 1.5 mL 2% BSA solution and resuspended with a pipet to generate single cells.
200,000 cells were spun down at 120g. the pellet was resuspended in 0.5 mL of Foxp3
fixation/permeabilization working solution (diluted 1:3, Invitrogen) and incubated at RT for 30 minutes. Cells
were washed in 1 mL 1X permeabilization buffer, centrifuged at 120g and resuspended in permeabilization
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buffer containing the antibodies in the respective contrentration. After 45 minutes incubation at room
temperature, the cells were centrifuged and resuspended in 200 µL permeabilization buffer. The cells were
transferred to one well of a 96 well plate with round bottom shape and washed three times by repeating the
centrifugation and resuspending step. The analysis was run at a calibrated flow cytometer (analyze 50.000 cells
at high speed in 150 µl buffer).
Supplementary Fig. 1
Acknowledgments
We would like to thank Dr. Feng Zhang for providing the plasmid pSpCas9(BB)-2A-Puro (PX459) V2.0; Cat. #
62988 for gene editing. We thank Nevena Stoynova and Mihaela Mate for cytogenetic technical assistance. We
thank the following agencies for financial support: The Danish Agency for Science, Technology and Innovation
(6114-00003B-768138), the People Programme (Marie Curie Actions) of the European Union’s Seventh
Framework programme FP7 under REA grant agreement (STEMMAD, grant No. PIAPP-GA-2012-324451),
Innovation found Denmark (BrainStem – Stem cell Centre of Excellence in Neurology, grant No. 4108-00008B).
The research leading to these results has received funding from the Innovative Medicines Initiative Joint
Undertaking under grant agreement number 115582 (EBiSC), the resources of which are composed of financial
contribution from the European Union’s Seventh Framework Programme (FP7/2007 -2013) and EFPIA
companies’ in-kind contribution.
References:
Chouraki, V., and Seshadri, S. (2014). Genetics of Alzheimer's disease. Adv Genet 87, 245-294. Conejero-Goldberg, C., Gomar, J.J., Bobes-Bascaran, T., Hyde, T.M., Kleinman, J.E., Herman, M.M., Chen, S., Davies, P., and Goldberg, T.E. (2014). APOE2 enhances neuroprotection against Alzheimer's disease through multiple molecular mechanisms. Mol Psychiatry 19, 1243-1250. Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A., Okamoto, S., Hong, H., Nakagawa, M., Tanabe, K., Tezuka, K., et al. (2011). A more efficient method to generate integration-free human iPS cells. Nat Methods 8, 409-412. Rasmussen, M.A., Holst, B., Tumer, Z., Johnsen, M.G., Zhou, S., Stummann, T.C., Hyttel, P., and Clausen, C. (2014). Transient p53 suppression increases reprogramming of human fibroblasts without aff ecting apoptosis and DNA damage. Stem Cell Reports 3, 404-413.
Table 1: Summary of lines
iPSC line names
Abbreviation in figures
Gender Age Ethnicity Genotype of locus
Disease
BIONi010-C-6 APOE-ε2/ε2 Male 18 African APOE-ε2/ε2 N/A
BIONi010-C-2 APOE-ε3/ε3 Male 18 African APOE-ε3/ε3 N/A
BIONi010-C-4 APOE-ε4/ε4 Male 18 African APOE-ε4/ε4 N/A BIONi010-C-3 APOE-KO Male 18 African APOE-knock-out N/A
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Table 2: Characterization and validation
Classification Test Result Data
Morphology Photography Visual record of the lines: normal iPSC morphology
Figure 1A
Phenotype Qualitative analysis by ICC
All lines express the stem cell markers Nanog, Oct4, SSEA3 and Tra-1-81
Figure 1B
Quantitative analysis by flow cytometry
Oct4, Sox2: more than 96 % positive for all lines (negative control: same line without antibody) SSEA1: less than 0.11% positive for all lines (negative control: same line without antibody)
Figure 1C
Genotype Karyotype (G-banding) and resolution
All lines show 46XY, Resolution 450-500
Figure 1E
Identity Microsatellite PCR (mPCR) OR STR analysis
The following markers were positive in all lines: Ectoderm: Sox1/Pax6 (all >25.5%); Mesoderm: CD34/CD56 (all >11.2%); Endoderm: CD184/Sox17 (all >48.5%)
Figure 1D
Donor screening HIV 1 + 2 Hepatitis B, Hepatitis C
Negative Data not shown but available with author
Genotype additional info (OPTIONAL)
Blood group genotyping N/A N/A
HLA tissue typing N/A N/A
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Table 3: Reagents details
Antibodies used for immunocytochemistry/flow-citometry